CA1277100C - Glycopeptides, process for their preparation and their use - Google Patents

Abstract

Abstract of the disclosure:

Compounds of the general formula I, II or III

are described, in which R1 is a hydrogen atom, an alkoxycarbonyl or arylalkoxy-carbonyl group conventionally used for protecting amino groups, or CH3-(CH2)m-CO, in which m =
0-16, R2 is -(CH2)n- or -(CHOH)n-, in which n = 1-10, R3 is a hydroxyl group, an O-alkyl or O-arylalkyl pro-tecting group, a group which activates carboxyl groups, a cephalin radical, an amino acid radical of oligopeptides, polypeptides or proteins which carries NH groups, or a carrier, A1, A2, A3 or A4 is a bonding dash or an amino acid radical present in glycophorin A, such as Ala, Val, Leu, Ile, Ser, Pro, Glu or Arg, in which, if appropriate, the reactive groups not forming part of the peptide bond are protected by protecting groups, and the radical T is T
in which R4 denotes a hydrogen atom or an acyl protecting group, R5 denotes a hydrogen atom, an acyl protecting group or the 2,3,4,6-tetra-O-acetyl-beta-D-galactopyranosyl or beta-D-galactopyranosyl radical, R6 denotes N3, HH2 or NHAc and R7 denotes H or CH3, together with a process for their preparation and their use, bound to a carrier, as artificial antigens, glyco-lipids or immunoadsorbents.

Description

84/B 020 - Ma ~89 The invent;on relates to compounds of the general formula I, II or III
R1-A1-T-N~-R2-CoR3 R1_A1_T_A2_NH_R2_coR3 II
R1-A1-T-A2-A3-T-A4-NH-R2-coR3 III
in ~h;ch R1 denotes a hydrogen atom, an alkoxycarbonyl or aryl-alkoxycarbonyl group convent;onally used for pro-10 tecting amino groups, or CH3-~CH2)m-C0, in ~hich m = 0-16, R2 denotes -~CH2)n- or -tCHOH)n-, ;n wh;ch n = 1-10, R3 denotes a hydroxyl group, an 0-alkyl or 0-arylalkyl protect;ng groupO a yroup uh;ch act;vates carboxyl 15 groups, a cephalin radical, an amino ac;d radical of ol;gopept;des, polypept;des or prote;ns ~hich carrles NH groups~ or a carrier, A1, A2~ A3 or A4 denotes a bonding dash or an amino acid radical present in glycophorin A, such as Ala, Val, Leu, lle, Ser, Pro, Glu or Arg, in ~hich, if appropriate, the reactive groups not forming part of the peptide bond are protected by protect;ng groups, and the rad;cal T d~notes R oJ --o R ÇH T

~CH
--HN ~CO--;n which R4 is a hydrogen atom or an acyl protectlng group, R5 ;s a hydrogen atom, an acyl protecting group or the 2,3,4,6-tetra-0-acetyl-beta-D-galactopyranosyl or beta-D-galactopyranosyl radical, 0 R6 ;s N3, NH2 or NHAc and ~l2~7~

R7 ;s a hydrogen atom or a methyl group, and also to a process for their preparation and their use, bound to a carr;er, as artificial antigens~ glycolip;ds or immunoadsorbents.
The treatment of erythrocytes of the M or ~ blood group with neuraminidase involves exposing the so-called Thomsen-Friedenre;ch ~T) antigen. Th;s T ant;gen has been characterized as the disaccharide unit -D-Gal-~1-3)-alpha-D-GalNAc, which is incorporated in the polypeptide cha;n of prote;ns v;a L-ser;ne or L-threon;ne radicals.
The TN ant;gen is obtained by cLeavage of the -0-glyco-s;d;cally bonded galacto un;t.
The T or TN antigen structures are present in, for example, the major glycoprotein of the erythrocyte membrane - namely the glycophorin - as "cryptoant;gens".
However, in the tumor t;ssue of various types of cancer, Springer ~Naturw;ssenschaften ~1983) 70, 369) has also been able to detect T and TN ant;gen structures, so these are to be regarded as tumor-assoc;ated ant;gens.
Although the carbohydrate part of the T and TN
ant;gen structures is character;zed, the oligopeptide part ;s not.
Uhlenbruck et al., Immunbiol. ~1983)~ 165~ 147, have proved that a glycopept;de structure ;s respons;ble for the spec;ficity of the T determ;nant. As glycopoly-pept;des isolated from glycophorin were used for th;s work, the T and TN spec;ficity could not be explained.
Through the chem;cal synthes;s of relevant glycopept;des whose serological and ;mmunochem;cal propert;es are com-parable to those of the natural T-specific structures, it would be possible to charactèrize the complete structure of the Thomsen-Fr;edenre;ch determ;nant and the T-specif;c tumor-associated determinant.
Glycopeptide inh;bitors, art;fic;al antigens or ;mmunoadsorbents w;th structures of the T-specif;c and TN-spec;f;c determinants are prepared us;ng chem;cal compounds ~ith these structural features, which can be reacted with suitable carr;er molecules. It ~ould be poss;ble to use synthetic T-spec;f;c and TN-sPecific antigens to obtain corresponding antibodies. These specific antibodies couLd be used for the diagnos;s and early detection of cancer.
The preparat;on of gLycopept;des or synthetic antigens is usually accompanied by the d;ff;culties assoc;ated ~;th the protecting group chemistry of carbo-hydrates and pept;des. Although Ferrar; and Pav;a tCarbohydrate Research ~1980), 79, C1-C7) ~ere able to synthes;ze an alpha-D-GalNAc-L-ser;ne or L-threon;ne seg-ment, attempts to remove a methyl ester protect;ng groupwere unsuccessful, mak;ng th;s segment unsu;table for the preparat;on of glycool;gopept;des. Although Paulsen ~Chem;cal Soc;ety Rev;e~s t19~4), Volume 13, No. 1, 15) and S;nay et al. ~Carbohydrate Research ~1983)~ 116/2 C9) were able to synthesize terminal glycopept;des as segments of the glycophor;n, these glycopept;des do not correspond to the T and TN spec;f;city, as Uhlenbruck ~as able to prove.
Surpr;s;ngly, it has now been sho~n that the un-protected glycopeptides of the formulae I and II~ andespec;ally of the formula III, have serolog;cal T or TN
act;v;ty ~h;ch ;s comparable to asialoglycophorin. It has also been sho~n, surpr;s;ngly, that glycopeptide haptens ~hich contain t~o ester groups can be coupled ~;th carrier molecules select;vely v;a the carboxyl group of the spacer. This opens up a ne~ route to the prepara-tion of complex glycopept;de ant;gens.
The object of the present invent;on was to prepare glycopeptides capable of coupling, from ~hich artificial T-active and TN-active antigens, ;mmunoadsorbents or glycollp;ds can be prepared by be;ng covalently bonded to carriers. Th~s object ~s ach;eved by prepar;ng compoun~s of the aeneral formulae I~ II and III, def;ned as ~nd~cated above, and b;nding them to carriers.
Preference ;s g~ven to compounds ~h;ch are present as part;al structures ;n glycophor;n~ of the formula V R1-Leu-T-NH (CH2) 5-CoR3 formula VI R1-Ser-T-NH (CH2) 5-COR
formula VII R1-Ile-I'-Ser-NH (CH2) 5-CoR3 formula VIII R1-Pro-T-Ala-NH(CH2~5~COR3 formula IX R1-Val-T-Glu(R9)-NHtCH2~5-COR3 formula X R1-Ile-T-Val-NH(CH2)5 CoR3 formula XI R1-Arg(R1U)-~-Val-NH(CH2)5-COR3 formula XII R1-Ala-T-Pro-Nt~ (CH2)5-CoR3 formula XIII R1-Val-T-Glu~R9)-Ile-T-Val-NH(CH2)5-COR3 formula XIV R1-Ile-T-Val-Arg(R10)-T-Val-NH(CH2)s~
CoR3 in ~hich R1 denotes a hydrogen atom or an acetyl group, R3 denotes a hydroxyl group, OCH3, O~tert~-butyl, O-benzyl, an active ester, a polylysine, protein or cephal;n rad;cal, a gel uhich carr;es am;no groups~
or a carrier, 5 R9 denotes a hydrogen atom or a protecting group for carboxyl groups, such as the methyl, benzyl or tert.-butyl group, R1D denotes a hydrogen atom or a protecting group for am;no groups, such as the NO? group, and 0 T has the mean;ng g;ven above.
In the process for the preparat;on of one of the compounds of the formulae I~ II and III, a) a compound of the general f-ormula XV

R~3C H2 RO)~O
ORs ~ xv ~ ~7 R0 ÇH
,CH

;n ~h;ch R4 denotes an acyl protect;ng group, preferably an acetyl or benzoyl group, R5 denotes an acyl protecting group or a 2,3,4,6-tetra-O-acetyl-beta-D-galactopyranosyl radical, R6 denotes N3 or NHAc, R7 denotes H or CH3, ~2~

R8 denotes an alpha,alpha-dimethyl-3,5-dimethoxybenzyl-oxycarbonyl~ benzyloxycarbonyl or 9-fluorenyl methoxycarbonyl group and R11 denotes 0-CH2Ph, is hydrogenated ;n the presence of a hydrogenation catalyst such as palladium/charcoal, and in the presence of an organic solvent such as methanol, ethyl acetate or diethyl ether, at room temperature, and, if appropriate, the free alpha-amino group of the serine or threon;ne ;s blocked with an alkoxycarbonyl or arylalkoxycarbonyl protecting group, preferably D~Z, Z, BOC or Fmoc, thereby forming a compound o~ the general formula XV in which the radicals R4, R5, R6 and R7 retain the meanings given above, R8 ;s a D~Z, Z, BOC or Fmoc group and R11 is a hydroxyl group, b) the product of step a) is reacted, by a condensation process conventionally used in peptide chemistry, such as the dicyclohexylcarbodiimide or active ester process, or using succinimide esters or p-n;trophenyl esters or mixed anhydrides, with a compound of the general formula XVI, ~hich has a free amino group H-A-NH-R2-CoR3 XVI
in ~hich A denotes a bonding dash or an amino acid radical, preferably Ala, Val, Pro, Ser, ~benzyl)Ser, Glutgamma-tert~-butyl) or Glu(gamma-Bn), R2 denotes -~CH2)n-, in ~hich n = 1-10, and R3 denotes OCH3, O-benzyl or O-tert.-butyl, or with a compound of the general formula XVII
H-A2-A3-0-benzyl XVII
in which A ;s Ala, Val, Pro, Ser~ ~benzyl)Ser, Glu~gamma-tert.-butyl), Glu~gamma-methyl) or Glu(gamma-Bn) and A3 ;s a bonding dash or an amino acid radical Ile or tNo2'Ar9~
to give a compound of the general formula XV in which the radicals R4, RS, R6, R7 and R8 are unchanged and R11 denotes A-NH-R2-COR3 or A2-A3-0-benzYl~

, the radicals A, A2, A3, R2 and R3 being unchanged, c) the protect;ng group DDz, BOC, Z or Fmoc in a product of step b~ is selectively cleaved by hydrolysis or hydro-genolysis, in a manner known per se, and the intermediate formed, ~hich carries alpha-amino groups, is reacted ~ith a compound of the general formula XVIlI
R1-A1_oR12 XVIII
in ~hich R1 denotes an acetyl group or a protect;ng group such as the DDZ, ~OC, Z or Fmoc group, A1 denotes a radical of one of the amino acids Ala, Val, Ile, Pro, Arg or ~N02)Arg and R12 denotes a hydrogen atom or an active ester radical such as the N-succinyl;mide or p-nitrophenyl radical, by a condensation process conventionally used in peptide chemistry, to give a compound of the general formula I or II i n ~hich R1 is an acetyl group or a DDZ, ~OC, ~ or Fmoc group, R2 is -(CH2)n-, in ~hich n = 1-10, R3 ;s OCH3, û-benzyl or O-tert.-butyl~
A1 ;s one of the amino acid radicals indicated and T is a radical of the formula XV with the meaning indicated under process step a), R8 and K11 together being a bonding dash, or to give a compound of the general formula XIX
R1-A1-T-AZ-A3-O-benzyl XIX
in which R1, A1, A2, A3 and T have the last mean;ngs indicated, d) the benzyl group in the product of the general formula XIX is selectively cleaved by hydrogenolys1s and the product 1s reacted, by one of the condensation methods conventionally used in peptide chemistry, with a compound of the general formula XX
H_A3_T_A4_N~I_R2_coR3 XX
in which A3, A4, T, R2 and R3 have the last meanings men-~
tioned, to give a compound of the general formula III, e) the protecting groups in a product of step c) or d) are removed by processes convent;onaLly used in carbo-hydrate chem;stry or pept;de chem;stry and the free amino group on the galactosam;nyl radical is selectively acetylated, a product of the general formula I, II or III
be;ng formed ;n ~hich R1 denotes an acet~l group or a protect;ng group~
~Z denotes -(CH2)n-, ;n ~hich n = 1-10, R3 denotes a hydroxyl group, T denotes the radical mentioned at the outset, in wh;ch R4 ;s a hydrogen atom, R5 ;s a hydrogen atom or a -D-galactopyranosyl rad;cal, R6 ;s NHAc and R7 ;s H or CH3, and A1, A2, A3 and A4 have the last mean;ngs ment;oned, f) a product of step e) is reacted in a manner known per se, by a conventlonal condensation process, w;th a poly-mer;c compound wh;ch carr;es amino groups, or a carrier, such as polylys;ne, cephal;n, a prote;n or a gel, to g;ve a synthet;c glycol;p;d, antigen or immunoadsorbent, and g) if appropriate, the remain;ng protect;ng groups ;n a product of step f) are removed in a manner known per se, by deblocking processes convent;onally used ;n peptide chem;stry.
The glycopept;des and glycopept;de der;vat;ves of the general formulae I, II and III are prepared by a process convent;onally used ;n pept;de chem;stry, for example ;n Houben-Weyl, Methoden der organischen Chemie ~Methods of organ;c chem;stry), Volume XV/1 and 2.
The act~vat;on of the carboxyl group can be effected for example by converting the carboxyl group to an acld halide, an azide, an anhydrlde, an imidazolide or an act;vated ester such as the N-hydroxysuccinimide ester or the p-nitrophenyl ester.
The am;no group can be act;vated by convers;on to a phosph;tam;de or by the phosphorazo process.
The conventional processes for the abovement;oned condensat;on react;ons are: the carbod;im;de process, the azide process, the m;xed anhydr;de process and the act;vated ester process, as descr;bed in "The Peptides", .~ .

~7~
_ 9 _ Volume 1, 1965 (Academic Press~.
The reactive groups which are not intended to take part in the condensation reaction are protected by pro-tecting groups which can eas;ly be removed later, for example by hydrolysis or reduction. Examples of protec-t;ng groups are also described in "The Peptides".
It is particulàrly advantageous also to protect the hydroxyl group of the serine radical. Protecting groups in this context are the benzyl or tert.-butyl group. It is particularly advantageous also to protect the guanidine group of arg;nine. A conventional protec-ting group in th;s context is the nitro group.
Protection of the omega-carboxyl group of glutamic acid is also advantageous. Conventional pro-tecting groups in this context are a tert.-butylcarbonyl, benzyl or methyl group.
The protecting groups can be cleaved by conven-tional processes according to the particular group, for example with trifluoroacetic ac;d or by mild reduction, for example with hydrogen and a catalyst such as palladium, or with ~Br in glacial acetic acid.
The protecting groups on the carbohydrate segment can be cleaved by conventional processes according to the type of protecting group tAngew. Chem. (1982), 94, 18~; CarbohYdr. Res. ~1983), 116, C9-C12). The az;do group can be converted to an amino group by mild reduc-tion, for exampLe w;th hydrogen and a catalyst such as palladium, or with sodium borohydride in the presence of n;ckel~II) chloride, and the amino group is converted to the acetylamino group by acetylation, for example with acet;c anhydride in methanol. The 0-acyl protecting groups are advantageously cleaved ;n a basic medium, for example by means of sodium methylate in methanol or sodlum hydrox;de or sodium carbonate in methanol.
The glycopeptides of the formulae I, II and III
are converted on the carboxyl group of the "spacer portion", bY a varietY of customary processes, to active ester der;vatives such as the N-hydroxysuccinimide ester or the p-nitrophenyl ester, and coupled with a carrier possessing one or more amino groups. Here, the reactive groups of a glycopeptide of the formula I, II or III
which are not intended to take part in the condensation reaction are particularly advantageously protected by protecting groups. In particular, the alpha-amino group of the terminal amino ac;d of the glycopept;de should be protected by a protecting group such as the 9-fluorenyl-methoxycarbonyl ~Fmoc), alpha,alpha-dimethyl-3,5~di-methoxybenzyloxycarbonyl (DDZ) or tert.-butoxycarbonyl (POC) group. The protecting groups can easily be removed later, for example by hydrolysis or photolysis.
The bind;ng of haptens to carriers is described, for example, in German Patent 32 30 427. Examples of carriers are proteins, preferably human or bovine serum albumin, glycoproteins, polymers such as polylysine or poly~glycyllys;ne), activated gels carrying amino, glycidyl, 2-aminoethylamino or active ester groups, poly-saccharides or polysaccharide gels activated by cyanogen bromide, or lipids, preferably cephalins or am;nated phospholip;ds.
These glycopeptide derivat;ves bound to carriers can be used as artificial antigens, glycol;p;ds or immunoadsorbents.
The following should be noted with respect to the var;ous abbreviat;ons used in the description, the examples and the cla;ms:
I. If no optical configuration is given for amino acid radicals, the L form is intended.
II. The following abbreviations are used to denote pro-tect~ng groups or activating groups:Adoc = adamantyloxycarbonyl Fmoc = 9-fluorenylmethoxycarbonyl DDZ = alpha,alpha-dimethyl-3,5-dimethoxybenzyloxy-carbonyl Z = benzyloxycarbonyl ~OC = tert.-butoxycarbonyl Butt = tert.-butyl Me = methyl OPN = p-nitrophenyl Bn = benzyl Bz = benzoyl Ac ~ acetyl DDBn = alpha,alpha-d;~ethyl-3,5-dimethoxybenzyl Gal = D-galactopyranosyl GalNAc = N acetyl-D-galactopyranosyl The structure of the follo~ing compounds ~as determined by 1H and 13C NMR spectroscopy and ~R
spectroscopy and also by means of elemental analysis.
The opt;cal rotations ~ere also determined.
The course of the reactions and the resulting products ~ere investigated by thin layer chromatography and by the HPLC technique.
The examples which follo~ illustrate the invention in greater deta;l ~ithout thereby implying a limitation.
Exa~ple 1 Preparat;on of compounds of the general formula I
R1-A1-T-NH-(CH2)5-CoR3 Scheme 1 belo~ describes the preparation of Com pounds 7 and 8. The general procedures are described in the experimental section uhich follo~s.
N-Acetyl-L-alanyl-(0-(2-acetamido-2-deoxy-alpha D-galactopyranosyl)-L-threonyl-N-(5-methoxycarbonyl)-n-pentylamide (Compound 7~
L-Alanyl-~0-(2-acetamido-2-deoxy-alpha-D-galactopyranosyl)-L-threonyl-N-~5-methoxycarbonyl~-n-pentylamide ~Compaund 8) The follo~ing compounds of the formula I ~ere prepared start;ng from a compound of the general formula XV as sho~n 1n scheme 1.

Compound No. R1 A1 T ~3 . ~
9 H Leu ~-GalNAc-Sf,~r OHn Ac Leu ~-GalNAc-Ser O~n 11 H Leu ~-Gal(1-3)-~-GalNAc-Ser OCH3 12 Ac Leu p-Gal~1-3)-~-GalNAc-Ser OCH3 13 H Ser p-Gal(1-3)-~-GalNAc-Ser OCH3 14 Ac Ser p-GaL(1-3)-~-GalNAc-Ser OCH3 ~2~
- 12 - .
Scheme 1 (GP = GeneraL Procedure) TlN3 TlN3 ¦ l.GP 7,8 1 .
~-Thr-O-Bn 2.GP 2 ~ DDZ-Thr-O-Active ester 3.GP la compound 1 co~pound 2 -TlN3 E~2N-(cH21s-coocH3 DDZ-Thr-NH-~CH2)5-COOCH3 GP lb compound 3 Tl N3 1. GP 6 H-Thr-NH- ( CH2 ) s-COOCH3 compound 4 +Ac-Ala / \ ~BOC-Ala GP 1 / \ GP

Ac-Ala-Thr-NH-~CH2)s-COOCH3 BOC-Ala-Thr-NH-~CH2)s-~OOCH3 compound 5 co~pound 6 _ _ 1. GP 11 GP 12 ~ ~ Tl ~ NHA C

Ac-Ala-Tt-NH- ~ CH2 ) s-COOCH3 H-Ala-Thr-NH- ~ CH2 ) s-COOCH3 u~ound 7 compound . _ ..
N3 ~ 0- ~3,4,6-t ri-0-a cetyl-2-azi do-2-deoxy-a lpha-D-ga la ct opy ranosyl) T NHAc = 0- ~2-a cetami do-3,4,6-t ri -0-a cetyl ~2-deoxy-alpha-D-galactopyranosyl) ~: .

~;~7~

Tt 0-(2-acetan;do-2-deoxy-alpha-D~-galactopyrano-syl)-L~threonyl Example 2 Preparation of compounds of the general formula II
R1 _A1 T-A2-NH- ~CH2~ 5-CoR3 II
Scheme 2 belo~ describes the preparation of Compounds 21 and Z2.
L-Isoleucyl-~0-(3-0-(Z,3,4,6-tetra-0 acetyl- -D-galacto-pyranosyl)-2-acetam;do-4,6-di-0-benzoyl-2-deoxy-alpha-D-galactopyranosyl)-L-seryl-L-valyl-N-~5-benzyloxycar-bonyl)-n-pen~ylamide (Compound 21) N-Acçtyl-L-;soleucyl-~0-(2-acetamido-2~deoxy-3-0-( -D-galactopyranosyl)-alpha-D-galactopyranosyl)-L-valyl-N-(5-benzyloxycarbonyl)-n-pentylam;de (Compound 22) The follo~;ng compounds of the formula II uere prepared start;ng from a compound of the general formula XV as shoun in scheme 2.

Compound No~ R1 A1 T A R3 23 Ac Ile d-GalNAc-Ser Ser OtH
24 Ac Ile p-Gal(1-3)-~-GalNAc-Ser Ser OCH
Ac Pro ~-Galtl-3)-~-GalNAc-Thr Ala OBn 26H Val ~-Gal(1-3)-~-GalNAc-Ser Glu(~-But ) OBn 27Ac Val ~-Gal(1-3)~-GalNAc-Ser Glu(~-But ) OBn 28~ Ac Val p-Gal(1-3)-~-GalNAc-Ser Glu OBn 29Ac Arg ~-Gal(1-3)-~-GalNAc-Thr Val OCH
Ac Ala ~-Gal(1-3)-~-GalNAc-Thr Pro OBn _c h em e 2 .:

T2NHAc T2NHAC
I l.GP 7,B
5 Z-Ser-O-Bn 2~GP 2 DDZ-Ser-O-Active ester 3.GP la compound 15 compound 16 T2NHAc ïo ~H-Val-NH-(CH2)$-COOBn DDZ-Ser-Val-NH-(CH2)s-COOBn ~, GP 1~
compound 17 T2NHAc H-Ser-Val-NH- ~ CH2 ) ~,-COOBn co~pound 18 ~BOC-Ile-OH f \ +Ac-Ile-OH
20GP l ~ ~ GP

1 2NHA~: T2NHAC
BOC-Ile-Ser-Va1-NH-(CH2)s-COOBn Ac-Ile-Ser-Val-NH-(CH23s-COOBn GP g comp 9 compound 20_ T 2 NHF c H-Ile-Ser-Val-NH-(CH2)s-COOBn Ac-Ile-Tq-Va1-NH-tCH2)s-t~OBn compound 21 compound 22 _ _ y T2NHAc = O~t3-û-t2~3,4,6-tetra-0-acetyl- -D-g3lacto-pyranosyl)-2-acetam1do-4,5-d~-0-benzoyl-2-deoxy-alpha-D-galactopyranosyl) Ts = 0-~2-acetamido-2~deoxy-3-û-t -D-galactopyrano-.
syl)-alpha-D-~alactopyranosyl)-L-seryl Example 3 Preparation sf compounds of the general formula III
R~-A1-T-A2-A3-T-NH-tCH2)5-CoR3 III
Scheme 3 below describes the preparat;on of Compound 36.
N-Acetyl-L-valyl-tO-t2-acetam;do-2-deoxy-3-0-~ -D-galacto-pyranosyl)-alpha-D-galactopyranosyl)-L-seryl-L-glutamyl-~gamma-~ert~-butyl)-L-;soleucyl-~0-~2-acetam;do-2-deoxy-3-0)-~ -D-galactopyranosyl)-alpha-D-galactopyranosyl))-L-seryl-L-valyl-N-~5-benzyloxycarbonyl)-n-pentylamide ~Compound 36) The follow;ng compounds of the formula III were prepared starting from a compound of the general formula XV as shoun ;n sche~e 3.

Compound No. R1 A1 T A2 A A R3 37 Ac Val ~-Gal(1-3)-~-GaLNAc Glu Ile Val OBn 38 Ac Val ~-Gal(1-3)-~-GalNAc Glu(~- Ile Val Oan Butt) and N-acetyl-L-;soleucyl-~0-~2-acetam;do-2-deoxy-3-0-~ -D-galactopyranosyl)-alpha-D-galactopyranosyl)-L-seryl-L-valyl-L-arg;nyl-~O-t2-acetam;do-2-deoxy-3-0)-~ -D-galacto-pyranosyl)-alpha-D-galactopyranosyl))-L-threonyl-L-valyl-N-~5-benzyloxycarbonyl)-n-pentylamide ~Compound 39) Scheme 3 12NHAc GP 7,8 T2NHAC
5 Z-Ser-O-Bn ~ H-Ser-OH
compound 15 ~

~2~RAC
I

10 Ac-Val-O-ACtive ester Ac-Val-Ser-OH

GP 1 b compound 3 2 H-Glu~-Butt~ O-Bn Ac-Val-Ser-Glu-ty~Butt)-O-Bn GP 1 compound 33 T2NHAc I

1- GP 7t Ac-Val-Ser-Glu~-Butt)-O-Active ester 2. GP la compound 34 T2NHAc +H-Ile-Ser-Val-NH-~CH2)s-COOBn GR lb c~o~ ~ r 12NHAc jr2NHAc ~ c-Val-Ser-Glu~-Butt)-Ile-Ser-Val-NH-~CH2)s-COOBn com~ u,nd 35 Ac-Val-Ts-Glu~r-Butt)-Ile-T9-Val-~-~CH2)s-COOBn compound 36 ~l2~

T NHAc = 0-(3-0-(2,3,4,6-tetra-0-acetyl- -D-galacto-pyranosyl)-2-acetamido-4~5-di-0-benzoyl-2-deoxy-alpha-D-galactopyranosyl) Ts = 0-(2-acetamido-2-deoxy-3-0-t ~D-galacto-pyranosyl)-alpha-D-galactopyranosyl)~L-seryl Example 4 Immob;l;zat;on of glycopeptide compounds on am;nated carriers The Compounds 7, 12, 14, 23 and 24 described in Examples 1, 2 and 3, wh;ch are ;n the form of methyl esters, were converted to carboxyl der;vatives according to GP 15. The Compounds 10, 22, 25, 27, 30, 36 and 38 described in ExampLes 1, 2 and 3, which are in the form of benzyl esters~ were converted to carboxyl derivatives according to GP 7 us;ng a polar solvent such as water, methanol or ~ethanol/ethyl acetate.
The result;ng glycopeptide derivatives, which have a free carboxyl group, were coupled ~ith aminated carriers according to ~P 16 and, ;f appropr;ate, the rema7ning protecting group was cleaved from these products.
The convent;onal coupling methods are described in German Patent Document A1-32 20 426.
Exp erimental sect;_on General Procedures tGP) ~ _ : Preparat;on of the peptide bond a) Preparation of an "active ester"
The carboxylic acid derivative (3 mmol)~ which has only one free carboxyl group and ;n ~h;ch the other re-active groups are protected, was dissolved in 50 ml of dry acetonitrile. N-Hydroxysucc;nimide (3 mmol) and dicyclohexyldiimide (3 mmol) were added w;th stirriny.
After 24 hours, the reaction mixture ~as filtered at 0C
and the filtrate was concentrated in vacuo. The result;ng syrup was used in the next reaction step without further purif;cation.
Commonly used solvent systems for thin layer chromato-graphy: chloroformtmethanol 9:1, 7:1, 3:1 and 1:1;
chloroform/ethyl acetate 1:1.

~2~

b) Preparation of a peptide bond The amino component t3 mmol), which, apart from a ~ree amino group, has the other reactive groups pro-tected, was dissolved ;n dry chloroform t25 ml) and adjusted to pH 9 with about 3 mmol of 4-tN,N-dimethyl-amino)pyridine, with stirring. After 10 minutes, the "active ester" from step a), dissolved in 25 ml of dry chloroform, was added. After 24 hours, the reaction mixture was extracted once by washing w;th 5X citric acid, dried with sodium sulfate and concentrated in vacuo. The resulting syrup was purified by column chromatography on silica gel.
Commonly used solvent systems for chromatography:
chloroform/ethyl acetate 1:1; chloroform/acetone 7:1, 4:1 and 1:1; chloroform/methanol 9:1 and 5:1.
GP 2: ~locking and protection of the alpha-amino group of the amino acid derivatives with the DDZ protecting group The DDZ protecting group was introduced into the amino acid derivatives according to a procedure described by Ch. Birr in Int. J. Peptide Protein Res. (1979) 13, 287-295.
GP 3: Blocking and protection of the alpha-amino group of the amino acid derivatives with the BOC protecting group The BOC protecting group was introduced into the amino acid derivatives according to the procedures des-cribed in Houben-Weyl, Methoden der organischen Chemie tMethods of organic chem;stry), Volume XV/I and II, publ;shed by E. Muller and E. Wunsch, 1974.
G _ : ~locking and protection of the carboxyl or alcoholic hydroxyl group of the am;no ac;d der;vatives with the tert.-butyl protecting group The tert.-butyl protecting group was introduced into the amino ac;d derivat;ves accordlng to the pro-cedures descr;bed in Houben-Weyl, Methoden der organischen Chem;e tMethods of organ;c chem;stry), Volume XV/I and II, published by E. Muller and E. Wunsch, 1974~

GP 5: Blocking and protection of the carboxyl or alcoholic hydroxyl group of the amino acid derivatives ~ith the D~Bn protecting group The DDBn protect;ng group was bonded to the car-boxyl or alcoholic hydroxyl group of the amino acidderivatives ;n the same manner as described in GP 4.
GP 6: Select;ve hydrolytic cleavage of the alpha,alpha-dimethyl-3,5-d;methoxybenzyloxycarbonyl (DDZ) protect;ng group from the glycopeptide The glycopeptide carrying the DDZ protecting group t3 mmol) was dissolved in 50 ml of 5X by volume trifluoroacet;c ac;d in methylene chloride at room tem-perature. After stirring for 30 minutes, the solution ~as neutral;zed to pH 7 with N-methylmorpholine. The mixture was washed once with ice-water and then with dilute hydrochloric acid. The organic phase was dried with sodium sulfate and concentrated to a syrup in vacuo~
The product was in the form of the hydrochloride.
A check by thin layer chromatography ~as carried out ~ith the following solvent systems: chloroform ethyl acetate 1:1; chloroform/acetone 4:1 and 1:1;
chloroform/methanol 4:1 and 1:1.
GP 7: Selective hydrogenolytic cleavage of the benzyl protecting group on the glycopeptide The glycopeptide carrying the benzyl protecting group t3 mmol) was dissolved in 7û ml of dry ethyl acetate and hydrogenated for 2 hours in the presence of 3.5 9 of 10X palladium-on-charcoal. The mixture ~as then filtered, the materlal on the filter was rinsed and the filtrate was concentrated to a syrup in vacuo. The resulting product was purified by column chromatography.
Solvents for chromatography: chloroform/acetone 4:1 and 1:1; chloroform/methanol 5:1 and 1:1.
GP ~: Selective hydrogenolytic cleavage of the benzyl-oxycarbonyl tZ) protecting group on the amino acldderivative The product carrying the Z protecting group t10 mmol) ~as hydrogenated for 1 hour in 50 ml of a mixture of ethyl acetate and methanol ~1:1) in the presence of ~27~

3 9 of 10X palladium-on-charcoal. The mixture ~as then filtered, the material on the ~ilter was rinsed and the filtrate was concentrated in v~cuo. The resulting syrup was taken up in chloroform and washed w;th dilute hydro-chloric ac;d. The organ;c phase was dried w;th sodiumsulfate and concentrated to a syrup in vacuo. The product, which was in the form of ~he hydrochloride, was pur;fied by column chromatography on s;l;ca gel.
~ommonly used solvent systems for chromatography:
chloroform/acetone 4:1; chloroform/methanol 5:1~
GP 9: Select;ve hydrolyt;c cleavage of the tert.-butoxycarbonyl ~OC) protecting group The product carry;ng the BOC protecting group ~2.6 mmol) was d;ssolved in 10 ml of 1.2 normal hydrogen chlor;de ;n dry glacial acetic acid at room temperature.
After st;rring for 20 minutes, the reaction mixture was treated ~;th 2~ ml of ether and concentrated ;n vacuo.
The resulting syrup was treated several t;mes ~;th toluene and concentrated unt;l the smell of acet;c acid was no longer detectable. The result;ng homogeneous product was pur;fied by column chromatography on silica gel.
Commonly used solvent systems for chromatography:
chloroform/acetone 9:1 and 4:1; chloroform/methanol 9:1 and 5:1.
GP 10: Select;ve hydrolyt;c cleavage of the alpha,alpha-d;methyl-3,5-dimethoxybenzyl (DDBn) group w;th tr;-fluoroacet;c ac;d a) The glycopept;de carry;ng the DDBn protect;ng group ~3 mmol) was dissolved in 50 ml of 5X by volume tr;-fluoroacet;c ac;d ;n methylene chlor;de at room tempera ture~ After st;rr;ng for 30 m;nutes, the solution was neutralized w;th N-methylmorphol;ne. The m;xture was washed once w;th ice-water and then with dilute hydra-chlor;c ac;d. The organ;c phase was dr;ed w;th sod;umsulfate and concentrated to a syrup ;n vacuo.
b) The DD~n protect;ng group can also be cleaved w;th ac;ds, such as HCl, ;n an aqueous med;um.
Solvent systems: chloroform/acetone 9:1, 4:1 and 1:1;

~2-7~
chloroform/methano~ 4:1 and 1:1.
_ 11: Selective reduction of ~he 2-azido group to the 2-amino group on the galactose unit with NiCl2/Na~H4 and subsequent acetylation of the amino group to the acetamido group The az;de compound (1 mmol) was dissolv~d ;n ethanol (5 ml) and NiCl2 solution (5 ml; 4X ~:v) of N;Cl2.6HzO in ethanol and 1X ~w:v) of added bor;c ac;d) and treated w;th NaBH4 (1 to 2 equivaLents).
After the react;on had ended, pyridine ~5 ml) and acetic anhydride (5 ml) were added and the mixture uas stirred at 20C for 3 to 24 hours. It was then concentrated and extracted by shak;ng with chloroform/water~ The organic phase was dried and concentrated in vacuo.
GP_12: Selective hydrogenolysis of the 2-az;do group to the 2-amino group on the galactose unit and subsequent acetylation of the am;no group to the acetamido group a) Reduction of the azido group to the amino group Hydrogen was passed for approx. 30 minutes into a 20 suspension of 500 mg of 10X palladium-on-charcoal and 100 ml of dry methanol. The p~ of the suspension was adjusted to 7 with a mixture of methanol and aqueous concentrated sodium carbonate solution (50:1). The azide compound ~5 mmol), dissolved in a small amount of methanol, was added and the mixture was then hydrogenated for 3 hours ;n the absence of light. ln the course of the hydrogenation, the pH was monitored and, if necessary, adjusted to 7. The mixture was then filtered, the material on the f;lter was carefully rinsed with methanol/ether and the filtrate was concentrated to a syrup in vacuo.
b) Acetylation The product from step a) was dissolved in dry methanol and treated w1th acetic anhydride ~50 mmol).
After stirring for 24 hours, the reaction mixture ~as concentrated in vacuo and the resulting syrup was treated several times with toluene and concentrated in vacuo.
The homogeneous product was purified by column chroma-tography on silica gel.

Commonly used solvent systems for chromatography:
chloroform/acetone ~:1, 5:1 and 2:1; chloroform/methanol 9:1 and 6:1.
In the acetylat;on, no~ only the amino group of the galactose unit was converted to the acetamide group, but also, ;f present, the alpha-amino group of the terminal amino acid on the glycopept;de.
GP 13: C(eavage of the 0-acyl protecting groups on the galactose un;t of the glycopeptide The glycopeptide ~2 mmol) was d1ssolved in 30 ml of methanol. An aqueous concentrated sod;um carbonate solution was added dropw;se to the methanol solut;on unt;l the pH was 11. After stirring for 20 hours, the reaction mixture was neutralized w;th act;vated ;on exchanger and f;ltered. The solution was concentrated to a syrup in vacuo and the rema;n;ng homogeneous product was purif;ed by column chromatography.
The cleavage of the acyl protecting groups can also be carr;ed out with catalyt;c quantities of sod;um methylate ;nstead of with sodium carbonate.
Commonly used solvent systems for chromatography:
chloroform/methanol/~ater 20:5:0.4 and 4:4:1.
GP 14: Cleavage of the ester-bonded gamma-tert.-butyl protecting groups on glycopeptides containing glutamic acid a~ The glycopeptide ~0.7 mmol), which contains gamma-tert.-butyl glutamate units, ~as dissolved in 4 ml of 90X by volume aqueous trifluoroacetic ac;d. After 50 m;nutes at 20C, 200 ml of diethyl ether were added and the precipi-tate was centrifuged off, washed w;th tw;ce 40 ml of d;ethyl ether and dried~
b) The cleavage o~ the gamma-tert.-butyl protect;ng group from glycopept;de segments containing gamma-tert.-butyl glutamate was carried out as described in GP 6.
Commonly used solvent systems for chromatography:
chloroform/methanol 5:1 and 3:1; chloroform/methanol ~ater 4:4:1.

5L;~7~
~3 -GP 15: Cleavage of the methyl ester on the spacer o~ the _ glycopeptide A solution of the methyl ester compound tO.5 mmol) in 30 ml of 1,4-dioxane/water (9:1) was saponified, ~ith S stirring, w;th 1 ml portions of a 1 normal sodium hydrox;de solution at 20C using thymolphthalein as the ;nd;cator, the consumption of alkali being monitored (about 0.5 mmol of NaOH). The mixture ~as neutral;zed with the ion exchanger Do~ex 50 WX-8H and filtered, the material on the f;lter was r;nsed and the f;ltrate was evaporated ;n vacuo. The remaining homogeneous product was purif;ed on a column of Sephadex G-Z5, us;ng methanol/water t1:1) as the eluent, and lyoph;l;zed.
Solvent systems for th;n layer chromatography: chloro-form/methanol/water 5:3:0.5 and 4:4:1.
GP 16: ~;nd;ng of glycopeptide haptens carrying a spacer group to aminated carr;ers The glycopeptide compounds carry;ng a spacergroup, and hav;ng a free carboxyl group capable of ~0 coupl;ng, were coupled by kno~n processes, either d;rectly w;th carbod;;m;des, for example 1-ethyl-3-~3 -d;methyl-am;nopropyl)carbod;;mide hydrochlor;de, or as act;vated es~ers, for example N-hydro~ysuccinimide derivatives, w;th prote;ns, for example bov;ne serum album;n, poly-pept;des such as polylysine, or aminated absorbents ascarr;er materials.
Ir, the case of products which may still carry a protected am;no ac;d un;t such as L-Glu(gamma-tert.-butyl~
or L-Glu(gamma-DD~n), the protect;ng group was cleaved by hydrolys;s in the presence of acet;c ac;d, trifluoro-acetic ac;d or an aqueous m;xture thereof.

Analyses Data:

Compound 3 lH-NMR = (400 MHz, CD30D) 4,78 (d, M-l J (1,2) = 3,4 Hz) 3,65 (s, CH30) 1,37 (d, CH3-Thr, J(CH3, CH) = 6,7 Hz Compound 4 (~)20 = ~ 97,7o (c = 1 in H2O) Compound 7 (~)20 = ~ 83 (c = 1 in H2O) lH-NMR (400 MHz, D2O) 3 = 4,85 (H-l, J(1,2) = 3,6 Hz) 3,60 (COOCH3) 1,9 (CH30) 1,32 (CH3-Thr, J(CH3, CH) = 6,6 Hz) Compound 11 ~ 79,2 (c = 1,2 ln H2O) lH-NMR (400 MHz, D2O) = 4,77 (d,H-l, J(1,2) = 3,2 Hz) 4,30 (d,H-l', J(1',2') = 7,7 Hz) 3,56 (s,COOCH3) 1,49-1,35 (CH2-Spacer) Compound 13 (~ )~ = + 83,2 (c = 1 in H2O) lH-NMR (400 MHz, D2O) = 4,72 (d,H-l, J(1,2) = 3,4 Hz) 4,27 (d,H-l', J(1',2') = 7,8 Hz) Compound 18 (dC) D = ~ 63,7 (c = 1 in Chloroform) lH-NMR (400 MHz, CDC13):
= 8,12-7,35 (m, Ph) 5,82 (d,NHAc, J(NH,2) = 9,0 Hz) 4,66 (d,H-l', J(1',2') = 7,9 Hz) 3,65 (CH30) 2,06-1,90 (5xAc) 1,62 and 1,32 (CH2-Spacer) Compound 19 (dC)~ = + 74,0 (c = 1 in CHCl3) lH-NMR 1270 MHz, CDC13):
= 3,66 (s, OCH3-Spacer) 4,25 (d, H-l' J(1',2') = 7,8 Hz) 4,82 (d, H-l, J(l,') = 3,6 Hz) 5,00 (dd, H-3') 5,10 (dd, H-2') 5,33 (dd, H-4') 5,92 (dd, H-4) ~77~
~ 2 Compound 22 (~ )~ = + 72,3 (c = 1 in MeOH/CHC13) Compound 23 (~)~ = + 98,3 (c = 1 in CH30H/CHC13 = 3:1) Compound 24 (~C)D = + 71,7 (C = 1 in CXC13/MeOH = 2:1) 13C-NMR (90 MHz, CDC13/CD30D 2:1) Cl'= 174-170 ~6xCO) 101,1 (C-l '; B) 99 ~ lC-1; ~C) 39,39 and 39,29 (CH2-Spacer) 36,89 (Ile) 33,91; 28,82 and 26,38 (CH2-Spacer) 22,15 (CH3 Acet) 15,27 (CH3 Ile) 10,89 (CH3 Ile)

Claims (13)

1. A compound of the general formula I, II or III

in which R1 denotes a hydrogen atom, a 9-fluorenyl-methoxycarbonyl (Fmoc), alpha, alpha-dimethyl-3,5-dimethyoxybenzyl-oxycarbonyl (DDZ) or tert.-butoxycarbonyl (BOC) group or CH3-(CH2)m-CO, in which m= 0-16, R2 denotes -(CH2)n- or (CHOH)n-, in which n = 1-10, R3 denotes a hydroxyl group, a benzyl or tert.-butyl group, a group which activates carboxyl groups, a cephalin radical, an amino acid radical of polylysine or bovine serum albumin, or a carrier, A1, A2, A3 or A4 denotes a bonding dash or an amino acid radical present in glycophorin A, and the radical T denotes T
in which R4 is a hydrogen atom or an acetyl or benzoyl group, R5 is a hydrogen atom, an acetyl or benzoyl group or the

2,3,4,6-tetra-O-acetyl-beta-D-galactopyranosyl or beta-D-galactopyranosyl radical, R6 is N3, NH2 or NHAc and R7 is a hydrogen atom or a methyl group.
2. A compound as claimed in claim 1 in which A1, A2, A3 or A4 denotes a bonding dash or an amino acid radical present in glycophorin A selected from Ala, Val, Leu, Ile, Ser, Pro, Glu and Arg.

3. A compound as claimed in claim 2 in which A1, A2, A3 or A4 denotes a bonding dash or an amino acid radical present in glycophorin A selected from Ala, Val, Leu, Ile, Ser, Pro, Glu and Arg in which reactive groups not forming part of the peptide are protected by a benzyl or tert.-butyl group, a nitro group, a tert.-butylcarbonyl group or a methyl group.

4. A compound of the general formula I, II or III, as claimed in claim 1, in which the compound is attached to a carrier that is a protein, a glycoprotein, a polymer, an activated gel carrying amino, glycidyl, 2-aminoethyl-amino or active ester groups, a polysaccharide or a poly-saccharide gel activated by cyanogen bromide or a lipid, so as to form an artificial antigen, a glycolipid or an immunoadsorbent.

5. A compound as claimed in claim 4 in which the protein is human or bovine serum albumin.

6. A compound as claimed in claim 4 in which the polymer is polylysine or poly(glycyllysine).

7. A compound as claimed in claim 4 in which the lipid is cephaline or aminated phospholipid.

8. A process for the preparation of a compound as claimed in claim 1, wherein a compound of the general formula XV

XV

in which R4 denotes a n acetyl or benzoyl group, R5 denotes an acetyl or benzoyl group or 2,3,4,6-tetra-0-acetyl-beta-D-galactopyranosyl, R6 denotes N3 or HHAc, R7 denotes H or CH3, R8 denotes an alpha,alpha-dimethyl-3,5-dimethoxybenzyl-oxycarbonyl, benzyloxycarbonyl or 9-fluorenyl-methoxycarbonyl group and R11 denotes O-CH2Ph, is hydrogenated in the presence of a hydrogenation catalyst, and in the presence of an organic solvent, at room temperature, and the product, which has a free carboxyl group, is reacted, by a condensation process conventionally used in peptide chemistry, with a compound of the general formula XVI, which has a free amino group in which A denotes a bonding dash or N2N-CH(R)CO-R2 denotes -(CH2)n-, in which n = 1-10, and R3 denotes OCH3, 0-benzyl or 0-tert.-butyl, or with a compound of the general formula XVII

H-A2-A3-O-benzyl XVII

in which A2 is Ala, Val, Pro, Ser, (benzyl)Ser, Glu(gamma-tert.-butyl), Glu(gamma-methyl) or Glu(gamma-Bn) and A3 is a bonding dash or an amino acid radical Ile or (NOz)Arg, to give a compound of the general formula XV in which the radicals R4, R5, R6, R7 and R8 are unchanged and R11 denotes A-NH-R2-COR3 or A2-A3-0-benzyl, the radicals A, A2, A3, R2 and R3 being unchanged, and the product which is formed after cleavage of the protecting group on the alpha-amino group of the serine or threonine unit is reacted with a compound of the general formula XVIII

in which R1 denotes an acetyl group or a 9-fluorenyl-methoxycarbonyl (Fmoc), alpha-dimethyl-3,5-dimethyoxybenzyl-oxycarbonyl (DDZ) or tert.-butoxycarbonyl (BOC) group or benzyloxycarbonyl (Z) R12 denotes a hydrogen atom or an active ester radical and A1 denotes a radical of one of the amino acids Ala, Val, Ile, Pro, Arg or (N02)Arg, by a condensation process conventionally used in peptide chemistry, to give a compound of the general formula I or II in which R1 is an acetyl group or a DDZ, BOC, Z or Fmoc group, R2 is -(CH2)n-, in which n = 1-10, R3 is OCH3, 0-benzyl or 0-tert.-butyl, A1 is a radical of one of the amino acids Ala, Val, Ile, Pro, Arg or (NO2) Arg and T is a radical of the formula XV in which R4 denotes an acetyl or benzoyl group, R5 denotes an acetyl or benzoyl group or a 2,3,4,6-tetra-0-acetyl-beta-D-galactopyranosyl radical, R6 denotes N3 or NHAc and R7 denotes H or CH3, and R8 and R11 together are a bonding dash, or is reacted with a glycopeptide compound which has only one free carboxyl group and which is formed by the hydrogenolysis of a benzyl ester compound of the formula XIX
R1-A1-T-A2-A3-O benzyl XIX
in which R1 is an acetyl group or a protecting group, A1, A2, A3 or A4 denotes a bonding dash or an amino acid radical present in glycophorin A selected from Ala, Val, Leu, Ile, Ser, Pro, Glu and Arg, and T is a radical of the formula XV in which R4, R5, R6 and R7 have the last meanings mentioned and R8 and R11 represent bonding dashes, by the condensation methods conventionally used in peptide chemistry, to give compounds of the general formulae I, II and III in which the radicals R1, R2, R3, A1, A2, A3, A4 and T have the last meanings mentioned.

9. A process as claimed in claim 8 in which the compound of the general formula XV is hydrogenated in the presence of an organic solvent that is selected from the group consisting of methanol, ethyl acetate and diethyl ether.

10. A process as claimed in claim 9 in which said hydrogenation catalyst is palladium/charcoal.

11. A process as claimed in claim 8 in which, after the hydrogenation step, the free alpha-amino group of the serine or threonine is blocked with an alkoxycarbonyl or arylalkoxy-carbonyl protecting group.

12. A process as claimed in claim 8 in which the product of the hydrogenation of the compound of the general formula XV
is condensed using a dicyclohexylcarbodiimide or active ester process, or using a succinimide ester or a p-nitrophenyl ester or a mixed anhydride.

13. A process as claimed in claim 8 in which R12 in formula XVIII is an N-succinimide radical or a p-nitrophenyl radical.